Genomic DNA Preparation From Mammalian Cells

(4-6 15cm plate)
Materials
  1. Four to six 15cm plates of 80-100% confluency monolayer cells
  2. PBS
  3. Lysis Buffer: T10E2N1507.5 solution containing 0.5% SDS and 0.5mg/ml proteinase K
  4. phenol
  5. CIA
  6. phenol/CIA
  7. Absolute ethanol (200 proof)
  8. 75% ethanol
  9. 0.5M NH4Ac
  10. T107.5 or T10E0.17.5
  11. RNase
  12. Restriction enzyme (eg. Xho I)
  13. Large tip pipet
  14. 50ml Falcon tube
  15. 50oC oven
  16. 37oC incubator
  17. LabQuake shaker
  18. Centrifuge
  19. Spectrophotometer
  20. Agarose gel aparatus and reagents
Procedure 
  1. Wash cells with PBS.
  2. Lyse cells in plate by adding 4ml T10E2N1507.5, 0.5% SDS, 0.5mg/ml proteinase K in each 15cm plate. swirl gently
  3. Transfer with large tip pipet to 50ml Falcon tube. Help with scraper, if necessary.
        Mix by gently inverting tube (avoid mechanical shearing of DNA).
  4. Incubate 2 hr at 50oC with occasional swirling. Then 37oC overnight (rotating wheel in an incubator).
  5. Extract with equal volumes of
    • Phenol (once)
    • Phenol/CIA  (twice)
    • CIA  (twice or until interphase is clear)
  6. by rotating tube on LabQuake shaker for 15 min and spin at 3000rpm at RT for 10 min and aspirate the top layer with large tip pipet to a new tube.
  7. 5. Add 2 volumes of cold ethanol and 0.5M NH4Ac and mix on rotating wheel. Spool precipitate with a sterile Pasteur pipette. Transfer precipitate to a fresh 50ml Falcon tube. wash the precipitate with cold 75% ethanol.
  8. Lyophilize or air dry overnight.
  9. Dissolve pellet with 1ml T107.5 or T10E0.17.5 by rotating at room temperature (may need 1 day or overnight).
  10. Add 1 ul RNase and incubate at 37oC for 30 min.
  11. Purify with Phenol/CIA-EtOH as in step 4.b(once)-5. Dissolve with 2ml T108.0.
       Check concentration by OD260 and Check quality by running agarose gel (+/- Xho I).
Hosted by www.Geocities.ws

1